This study was designed to assess the effect of S-nitrosoglutathione monoethyl ester (GSNO-MEE) a membrane-permeable analog of S-nitrosoglutathione (GSNO), on isolated rat heart during cardioplegic ischemia, and to monitor the release of nitric oxide from GSNO-MEE in isolated hearts using the endogenous myoglobin as an intracellular nitric oxide trap and the hydrophilic MGD-Fe2+ complex as an extracellular nitric oxide trap. During cardioplegic ischemia, GSNO-MEE (20-200 fmol/l) induced the formation of nitrosylmyoglobin (MbNO), which was detected by ESR spectroscopy. Inclusion of MGD-Fe2+ (50 fmol/l Fe2+ and 500 fmol/l MGD) into cardioplegic solution along with GSNO-MEE yielded an ESR signal characteristic of the MGD-Fe2+-NO adduct. However, MGD-Fe2+ trap did not prevent the formation of the intracellular MbNO complex in myocardial tissue. During aerobic reperfusion, denitrosylation of MbNO complex slowly occurred as evidenced by the decrease in ESR spectral intensity. GSNO-MEE treatment did not affect ubisemiquinone radical formation during reperfusion.